دورية أكاديمية
أعرض في EDS

Heterologous expression and characterization of plant Taxadiene-5α-Hydroxylase (CYP725A4) in Escherichia coli.

التفاصيل البيبلوغرافية
العنوان: Heterologous expression and characterization of plant Taxadiene-5α-Hydroxylase (CYP725A4) in Escherichia coli.
المؤلفون: Rouck JE; Department of Comparative Biosciences, Department of Biochemistry, Department of Bioengineering, Division of Nutritional Science, Center for Biophysics and Quantitative Biology, Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA., Biggs BW; Manus Biosynthesis, 1030 Massachusetts Avenue, Suite 300, Cambridge, MA 02138, USA; Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA., Kambalyal A; Department of Comparative Biosciences, Department of Biochemistry, Department of Bioengineering, Division of Nutritional Science, Center for Biophysics and Quantitative Biology, Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA., Arnold WR; Department of Comparative Biosciences, Department of Biochemistry, Department of Bioengineering, Division of Nutritional Science, Center for Biophysics and Quantitative Biology, Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA., De Mey M; Centre for Industrial Biotechnology and Biocatalysis, Ghent University, Coupure Links 653, B-9000, Belgium., Ajikumar PK; Manus Biosynthesis, 1030 Massachusetts Avenue, Suite 300, Cambridge, MA 02138, USA. Electronic address: pkaji@manusbio.com., Das A; Department of Comparative Biosciences, Department of Biochemistry, Department of Bioengineering, Division of Nutritional Science, Center for Biophysics and Quantitative Biology, Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA. Electronic address: aditidas@illinois.edu.
المصدر: Protein expression and purification [Protein Expr Purif] 2017 Apr; Vol. 132, pp. 60-67. Date of Electronic Publication: 2017 Jan 18.
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: Academic Press Country of Publication: United States NLM ID: 9101496 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1096-0279 (Electronic) Linking ISSN: 10465928 NLM ISO Abbreviation: Protein Expr Purif Subsets: MEDLINE
أسماء مطبوعة: Publication: Orlando, FL : Academic Press
Original Publication: San Diego : Academic Press, c1990-
مواضيع طبية MeSH: Cytochrome P-450 Enzyme System*/biosynthesis , Cytochrome P-450 Enzyme System*/chemistry , Cytochrome P-450 Enzyme System*/genetics , Cytochrome P-450 Enzyme System*/isolation & purification , Plant Proteins*/biosynthesis , Plant Proteins*/chemistry , Plant Proteins*/genetics , Plant Proteins*/isolation & purification, Alkenes/*chemistry , Diterpenes/*chemistry , Escherichia coli/*metabolism , Taxus/*genetics, Binding Sites ; Escherichia coli/genetics ; Kinetics ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/chemistry ; Recombinant Proteins/genetics ; Recombinant Proteins/isolation & purification ; Taxus/enzymology
مستخلص: Taxadiene-5α-Hydroxylase (CYP725A4) is a membrane-bound plant cytochrome P450 that catalyzes the oxidation of taxadiene to taxadiene-5α-ol. This oxidation is a key step in the production of the valuable cancer therapeutic and natural plant product, taxol. In this work, we report the bacterial expression and purification of six different constructs of CYP725A4. All six of these constructs are N-terminally modified and three of them are fused to cytochrome P450 reductase to form a chimera construct. The construct with the highest yield of CYP725A4 protein was then selected for substrate binding and kinetic analysis. Taxadiene binding followed type-1 substrate patterns with an observed K D of 2.1 ± 0.4 μM. CYP725A4 was further incorporated into nanoscale lipid bilayers (nanodiscs) and taxadiene metabolism was measured. Taxadiene metabolism followed Michaelis-Menten kinetics with an observed V max of 30 ± 8 pmol/min/nmol CYP725A4 and a K M of 123 ± 52 μM. Additionally, molecular operating environment (MOE) modeling was performed in order to gain insight into the interactions of taxadiene with CYP725A4 active site. Taken together, we demonstrate the successful expression and purification of the functional membrane-bound plant CYP, CYP725A4, in E. coli.
(Copyright © 2017 Elsevier Inc. All rights reserved.)
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معلومات مُعتمدة: R01 GM115584 United States GM NIGMS NIH HHS
فهرسة مساهمة: Keywords: E. coli; Nanodiscs; Recombinant expression; Taxadiene; Taxadiene-5α-ol
المشرفين على المادة: 0 (Alkenes)
0 (Diterpenes)
0 (Plant Proteins)
0 (Recombinant Proteins)
0 (taxa-4(5),11(12)diene)
9035-51-2 (Cytochrome P-450 Enzyme System)
تواريخ الأحداث: Date Created: 20170123 Date Completed: 20170703 Latest Revision: 20201209
رمز التحديث: 20240628
مُعرف محوري في PubMed: PMC5741078
DOI: 10.1016/j.pep.2017.01.008
PMID: 28109855
قاعدة البيانات: MEDLINE
الوصف
تدمد:1096-0279
DOI:10.1016/j.pep.2017.01.008